There are persistent questions regarding the evolution and function of the immune system. Previously studies performed in non-mammalian vertebrates have helped to reveal basic features of the immune system that are universal. In other cases, studies outside of mouse and human have underscored the great plasticity seen in certain genes/molecules/mechanisms related to immunity. In our studies of the immune system in cartilaginous fish (sharks, skates, and rays), the oldest animals with adaptive immunity, we have discovered a new immunoglobulin (Ig) isotype (the new antigen receptor or IgNAR) with a very unusual binding site composed of a so-called single-chain variable (V) region. This unusual antibody is of potential therapeutic/diagnostic interest, but for this proposal it has opened new avenues to examine antibody structure and function throughout evolution. Because of their atypical gene organization, we propose that the cartilaginous fish Ig genes are expressed by cells that are 'layered'in the immune system: 'innate'receptors with fixed specificities expressed first in development, followed by cells with rearranged IgV receptors expressed as multimeric IgM, followed by lymphocytes that produce monomeric IgM or IgNAR. We propose that the 'innate'receptors and 19S IgM form a first line of defense, and the 7S and IgNAR provide the highly specific humoral immunity. Unexpectedly, the NAR V domain is also used by a subset of ??? T cell receptors (TCR), and we propose that such receptors complement the canonical pool of ??? TCR, in the same ways that IgNAR is believed to complement IgM. Finally, an IgD homologue was discovered that was revealed to be an orthologue of a close cousin of IgNAR, IgW. The function of IgD in humans is not known, and we propose that an understanding its ancestry could be useful in unraveling its role in generating immune responses. Public information: Unusual animal models found at crucial steps in the evolutionary tree are used to help understand basic features of the human immune system-we use sharks, the oldest living animals with an adaptive immune system, and Xenopus, an amphibian at the transition point between aquatic and terrestrial vertebrates. We are especially interested in studying the structure and function of human immune molecules (the antibody isotype IgD and the ??? T cell receptor), whose functions have not been well defined.